Discovery and Structure-Based Design of Potent Covalent PPARγ Inverse-Agonists BAY-4931 and BAY-0069

The ligand-activated nuclear receptor peroxisome-proliferator-activated receptor-γ (PPARG or PPARγ) represents a potential target for a new generation of cancer therapeutics, especially in muscle-invasive luminal bladder cancer where PPARγ is a critical lineage driver. Here we disclose the discovery of a series of chloro-nitro-arene covalent inverse-agonists of PPARγ that exploit a benzoxazole core to improve interactions with corepressors NCOR1 and NCOR2. In vitro treatment of sensitive cell lines with these compounds results in the robust regulation of PPARγ target genes and antiproliferative effects. Despite their imperfect physicochemical properties, the compounds showed modest pharmacodynamic target regulation in vivo. Improvements to the in vitro potency and efficacy of BAY-4931 and BAY-0069 compared to those of previously described PPARγ inverse-agonists show that these compounds are novel tools for probing the in vitro biology of PPARγ inverse-agonism.

to clinical evaluation.To test the therapeutic hypothesis that inducing a repressive PPARg complex using an inverse-agonist might be clinically beneficial for patients, we set out to identify potent and selective inverse-agonists with favorable pharmacokinetic and pharmacodynamic properties.Here we report the discovery of new potent and efficacious PPARg covalent inverse-agonists.While these compounds are limited in their in vivo pharmacokinetic properties, they are valuable tools for in vitro studies.RESULTS AND DISCUSSION: A high-throughput screening campaign for PPARg inverse-agonists was undertaken as outlined in Figure 2A.A chemical library containing more than 4 million compounds was interrogated using an ultrahighthroughput PPARg Biochemical Competitive Binding Assay to identify binders of the PPARg ligand binding domain (LBD) that are competitive the fluorescent labeled PPARg ligand, fluormone™.Compounds that bind to the binding pocket of PPARg LBD lead to a decrease in TR-FRET signal between fluormone and labeled PPARg.Compounds were screened in duplicate at a single concentration (10µM).Candidate binders identified in the primary screen were validated by retesting in the PPARg binding assay and a total of 15,152 primary hits and 11,421 confirmed hits for an overall positive rate of 0.3% (Chart S1) for PPARg binders .
To deconvolute functional activity and prioritize confirmed binders, functional biochemical and cellular assays were performed at a single dose (10 µM) in a panel of assays.A PPARg:MED1 TR-FRET biochemical co-activator interaction assay was used to measure the effect of compounds on the interaction between PPARg LBD and a peptide containing the "LxxLL" nuclear receptor interaction motif from the co-activator, MED1 (TRAP220/ DRIP-205).A signal increase is indicative of a potential agonist and known PPARg agonist GW1929 12 responded accordingly.To investigate candidate inverse-agonists, the assay was adapted to use a peptide containing the interaction motif from the co-repressor, NCOR2 (Smrt ID2) 13 in place of MED1 peptide.Compounds that induce interactions between PPARg LBD and peptide from the co-repressor NCOR2 would be indicative of inverse-agonists.Known inverse-agonists T0070907 11 and SR10221 10 responded accordingly.
A large proportion of the initially confirmed HTS candidates did not show activity in the coregulator recruitment assays (bottom left quadrant of Fig 2B ) and are likely simple binders or neutral antagonists.Another large fraction overlapped in directionality of assay signal with the PPARg agonist, GW1929, in the upper left quadrant of Figure 2B.PPARg inverse-agonists T0070907 and SR10221 demonstrated a concentrationdependent increase in signal in the NCOR2 biochemical assay.A small number of candidates led to a signal increase in the co-repressor recruitment assay (lower right quadrant of 2B), indicating potential inverse-agonists.Interestingly, agonists led to a decreased signal in the inverse-agonist biochemical assay with NCOR2 peptide, and conversely, inverse-agonists decreased signal in the agonist assay with MED1 peptide.This indicates that inverse-agonists may induce a structural conformation that disrupts basal interactions between PPARg and coactivators, further shifting the equilibrium from activation to repression.Whether this also occurs in the cellular context is unclear.To eliminate possible assay artifacts and verify activity in a cellular context, high-throughput RT-qPCR monitoring the mRNA transcripts of the canonical PPARg target gene FABP4 was performed.The effects of compounds on the expression of FABP4 in RT112 cells were also monitored using a high-throughput nanoluciferase reporter assay (RT112-FABP4-NLucP 3 ) to evaluate and triage candidate compounds based on cellular activity with a single dose of 10µM.
Subsequent predictive filtering of chemical structures to eliminate undesirable structures refined the list to 561 preferred candidates for testing in dose-response format.Preferred hits were evaluated in a co-repressor recruitment TR-FRET assay measuring ligand dependent changes in the interaction between PPARg and a peptide from NCOR2, in addition to RT-qPCR of the canonical PPARg target gene, FABP4.Compounds were also tested in a previously described cellular reporter assay for PPARg transactivation, RT112-FABP4_NLucP reporter assay 3 .
To select the most suitable starting point for a candidate optimization effort, we started with an absolute requirement for confirmed binding with a dose-dependent increase in signal in the PPARg:NCOR2 interaction assay.Candidates were further triaged by a requirement for dose-dependent inverse-agonist activity in one or more of the cellular assays in PPARg-activated bladder cancer cell lines, including RT-qPCR in RT112 cells, or RT112-FABP4-NLucP.Five favored chemotypes were identified.One promising candidate, 6a, which shares the same chloro-nitro benzamide covalent warhead as T0070907 (Compound 4), was selected for optimization.concentration hit validation, and dose-response curves to identify candidate hit 6a.B. Hit validation comparing fluorescent signal from PPARg:MED1 co-activator recruitment and PPARg:NCOR2 co-repressor recruitment (CRR) LanthaScreen TR-FRET interaction assays for candidate compounds tested at 10µM.PPARg probe compounds were added in dose-titration for benchmarking assay performance.Probe compounds included GW1929 (agonist), SR10221 (inv agonist), and T0070907 (inv agonist).
To help guide medicinal chemistry efforts, 6a was co-crystallized with the PPARg LBD bound to a peptide derived from the co-repressor NCOR2 (Figure 3).6a covalently modified C313 of PPARg isoform 2, in a similar manner to T0070907 14 .Crystal structures of 6a and T0070907 show that Helix-12, an essential interaction surface for co-activator proteins, is sequestered into the canonical ligand binding site and is thus unavailable to recruit the co-activators (Fig 3B).The conserved residues of the hydrophobic receptor interaction motif (LXXIIXXXL) of NCOR2 are key to the interaction with PPARg bound to the inverse-agonists (Fig 3C).Intriguingly, the C-terminal residue (Y475 in PPARg isoform 1 NP_005028, or Y505 in PPARg isoform 2; NP_056953.2) interacts directly with the amide linker of the inverse-agonist.
Compared to known inverse-agonist T0070907, the lipophilic tail of 6a, consisting of the benzoxazole and 3-tolyl group, extends further towards the lipophilic co-repressor binding surface.The binding region is relatively narrow, with the potential to interact with solvent around the central benzoxazole ring.To systematically investigate the insights gleaned from this crystal structure, the structure activity relationship (SAR) for the compound was evaluated within 3 structural regions (Scheme 1), the NCOR-interacting ring, the potentially solvent-interacting core, and the covalent warhead.A variety of small substituents on the aryl ring at different positions showed strong preference for meta / para substitution (6a, 6c (BAY-4931)), while ortho substitution (6b) dramatically reduced potency and maximal efficacy in the cellular assays (Table 1).Incorporation of electron-poor rings (6d-e) significantly improved activity, though combining bulk at the para position with an electron deficient ring did not provide an additive effect (6f).Substituted 3-pyridines maintained activity in the co-repressor recruitment assay (6g-h) (Table1).
Functional activity was evaluated by testing candidate compounds in NCOR2 recruitment assay as well as the previously described RT112-FABP4-NLucP cellular reporter assay for PPARg transactivation 3 .Again, para substitution was strongly preferred (BAY-4931), while the combination of the 3-pyridyl and 4-ethyl substitutions led to favorable effects in cellular assays (6h).Notably, the variability in maximal efficacy (Emax) in the cellular assays was less pronounced than in the biochemical assay.To profile the antiproliferative effect of functional inverse-agonists, compounds were evaluated in UM-UC-9 bladder cancer cells, which possess a focal gene amplification of PPARG to greater than approximately 25 copies and are exquisitely sensitive to PPARg modulation 3 .Again, para substitution was strongly preferred, as only (BAY-4931) and (6h) demonstrated compelling antiproliferative potency and efficacy (Table 1).Interestingly, 6g maintained potency, but had poor efficacy in the UM-UC-9 proliferation assay indicative of a partial inverse-agonist.showing BAY-4931 is more potent and efficacious than 6a.The hypothesis that a stabilized binding mode of the inverse-agonist is important for increased efficacy fits with the finding that T0070907 exhibits conformational dynamics and a different binding mode when bound to PPARg prior to recruitment of NCOR2 as described by Shang et al 14 (PDB: 6C1I).The observed flipped binding mode of T0070907 does not allow for any interactions to NCOR2 and Helix-12 is also not sequestered (Figure S4).
Selected active compounds from Table 1 were profiled for metabolic stability, Caco-2 permeability, aqueous solubility, glutathione (GSH) stability and extent of covalent modification of the PPARg ligand binding domain (Table 2).All compounds tested were poorly soluble and highly metabolized in rat liver hepatocytes (≥ 70% of LBF in rats assuming 4.2 L/h/kg 15 ), but moderately stable in human liver microsomes (LBF in human of ~1.3 L/h/kg 16 ) (Table 2).Permeability ranged from low (≤ 10 nm/s, 6c) to favorable (≥ 70 nm/s, 6g), with none of the compounds tested showing signs of P-glycoprotein (P-gp) mediated efflux (Table 2).Despite improved human microsomal stability and permeability for pyridines 6g and 6h, these compounds did not lead to an improvement in rat hepatocyte stability or solubility compared to BAY-4931.Thus BAY-4931 was selected as the preferred compound for further optimization.We next turned our attention to the potentially solvent exposed central core to improve physicochemical and ADME properties.Compounds with variable central cores were synthesized according to Scheme 1b.
Inverting the benzoxazole regiochemistry (8a) maintained co-repressor recruitment, whereas making the benzoxazole the NCOR-interactor and the aryl the linker ablated co-repressor recruitment (8g, h) (Table 3).Benzannulated heteroaryl cores provided mixed results, as N-Me benzimidazoles reduced co-repressor recruitment (8b, c) while an imidazo-pyridine and a benzotriazole retained co-repressor recruitment (8d, e) (Table 3).Efforts to replace the amide linker with a sulfonamide failed to retain activity (8f).Modifications that retained biochemical activity resulted in unacceptable loss in efficacy and potency in the cellular assays.Despite the lack of improvement in cellular assays, selected compounds from Table 3 were profiled in tier 1 ADME to understand the impact of increased polarity on pharmacokinetic properties.Notably, the more polar N-heteroaryl cores failed to improve the solubility or metabolic stability of the parent compound, though 8c and 8d demonstrated improved permeability (Table 4).Since no modifications made thus far improved hepatocyte stability, compound 6a was subjected to metabolite identification studies.The sole identified metabolite of 6a results from GSH displacing the Ar-Cl warhead (Figure 5).Envisioning that improving the metabolic stability was essential to continue to progress the chemical series, we next modulated the reactivity of the covalent warhead.and its metabolite P-1 before (blue) and after 1 h incubation (red) in rat hepatocytes and structure proposal of the metabolite P-1 based on the exact masses of the metabolite P-1 and its MS-MS fragments Less-reactive warheads decreased activity in PPARg activity assays (Table 5).Changing the activating nitro group to a variety of less electron withdrawing groups (9a-d) caused significant or full activity loss, though nitrile 9d remained active in the co-repressor recruitment assay.Removing or positionally shifting the covalent warhead resulted in inactive compounds (9e, f).Warheads exploiting alternate chemistry, such as heteroarenes and acrylamides, were inactive (9g, h).Aryl halides maintained similar in vitro activity to the parent chloride BAY-4931 (9i-k).However, these substitutions failed to improve metabolic stability (Table 6).Despite poor aqueous solubility, low metabolic stability, and low permeability (see Table 6), chloride BAY-4931 and bromide 9j (BAY-0069) possessed the best overall in vitro profile; thus, both were characterized in vivo.Selectivity profile of BAY-4931 and BAY-0069: PPARG is a member of a family of lipid-activated nuclear receptors, which encompasses PPARA and PPARD, with additional high homology with PXR.As such, BAY-4931 and BAY-0069 were tested in cellular reporter activity assays for mouse Pparg to determine cross-species reactivity, as well PPARA, PPARD, and PXR to determine selectivity (Table 7).BAY-0069 only acted as an inverse-agonist at PPARg, with little activity at the homologous PXR.When tested for CYP inhibition across a panel of the most relevant CYP enzymes for drug-drug-interactions, both BAY-4931 and BAY-0069 only inhibited CYP2C8 (Table 7).In vitro cell profiling: Compounds BAY-4931 and BAY-0069 were compared to probe compounds, T0070907 and SR10221, in dose response across a variety of cell lines.These studies highlight subtle differences of the in vitro profiles of these newly described compounds (Figure 6A-E).A dose dependent increase in the interaction between PPARg and co-repressor peptides from NCOR1 and NCOR2 were observed as expected for inverse-agonists.A dose-dependent decrease in the interaction with peptide from co-activator MED1 was also observed, even in the absence of exogenous agonist, indicating a destabilizing effect of inverse-agonists on basal interactions between PPARg and co-activators.BAY-4931 and BAY-0069 led to antiproliferative effects in PPARG amplified cell line UM-UC-9, while agonist rosiglitazone led to a modest, but reproducible increase in proliferation in this cell line.
During kinetic proliferation profiling in sensitive bladder cell lines 3 , we noticed a delay of 2-5 days after initial dosing prior to observing changes in the proliferation rate and eventual cytostasis.Therefore, we tested the effects of T0070907 or BAY-4931 on proliferation in a 12-day multiplexed cell line panel screen to screen for potentially sensitive cell lines (PRISM, Broad Institute, Figure S1) 17 .Candidate cell lines from this study in addition to other PPARG-dependent cell lines from DepMap were selected for further evaluation in colony formation assays to enable extended treatment with compounds for 7-14 days (Figure 6F and Figure S2).We compared the antiproliferative effects of inverse-agonists T0070907 and BAY-4931 to the neutral antagonist GW9662.
At a concentration of 100nM, BAY-4931 shows antiproliferative effects across the majority of the cell lines selected for predicted sensitivity.At the same concentration, T0070907 also shows antiproliferative effects in these cell lines, but to a more modest degree.UM-UC-9, HuP-T4, and PaCaDD-188 were among the most sensitive cell lines in the colony formation assays treated with BAY-4931.BFTC905, a bladder cell line, was chosen as a control as it was not identified as sensitive to compounds in the PRISM panel or genetic perturbation of PPARG in DepMap and compounds had minimal effect; the lack of growth inhibition in BFTC905 by the inverse-agonists taken together with antiproliferative effects in the predicted sensitive cell lines suggests compound selectivity for the subset of PPARG-dependent cell lines.

In vitro effects on gene expression:
The effects of PPARg modulators on global mRNA regulation were evaluated by RNA sequencing in the RXRA p.S427F hotspot mutation bladder cell line, HT-1197 (Figure S3).Comparing treatment effects of inverse-agonists T0070907 at 500nM to BAY-4931 at 200nM, it was observed that the same genes are regulated by T0070907 and BAY-4931 with the same directionality of the effect.The genes expression effects anticorrelate with the effects of the PPARg agonist, rosiglitazone (Figure S3a).Furthermore, the entire gene set was regulated proportionately more with BAY-4931 (Figure S3b) under the conditions tested.This indicates that at maximal receptor occupancy, BAY-4931 drives the equilibrium more towards repression than T0070907, analogous to expected effects of a partial agonist compared to a full agonist.In vivo profiling: Based on the strong in vitro effects and high selectivity of BAY-0069 in addition to slightly improved microsomal stability over BAY-4931 (Table 6), we elected to profile BAY-0069 in vivo.Unfortunately, the low intrinsic solubility of BAY-0069 prevented profiling by i.v.administration; however, compound exposure was assessed by oral, intraperitoneal, and subcutaneous administration, all at 100 mg/kg (Figure 7).Despite the high dose administered, the obtained exposure was found to be very low by all three routes with i.p. showing the best AUC0-tlast of 0.26 h*mg/L and a Cmax of 59 nM (see Table S2).Corrected for protein binding, the unbound Cmax of BAY-0069 covered the IC50 from the FABP4-NLucP reporter gene assay but failed to exceed the antiproliferative IC50u from UM-UC-9 cell lines, though intraperitoneal administration came closest with a Cmax,u / IC50, u of 0.77.F. Colony Formation of FABP4 expression, comparable with SR10221, despite more robust in vitro inverse agonism in NCOR2 recruitment, RT112-FABP4-NLucP luciferase repression, and UM-UC-9 proliferation (Figure 8).Upregulation of ANXA3 expression was statistically significant by SR10221, but not by BAY-0069.Note that for BAY-0069, the maximum tolerable dose was reached and repeat dosing was not possible.

CONCLUSIONS:
Herein we disclose the discovery of a halo-nitroarene series of covalent PPARg inverse-agonists.Chemical optimization through structure-informed design led to discovery of a potent and selective PPARg inverse-agonists.The extended benzoxazole core brought a lipophilic arene in close contact with the lipophilic surface of the co-repressor NCOR2, while the binding of the chloro-nitroarene warhead sequesters Helix-12, an essential binding surface for the co-activator MED1, deep inside the protein.
Target engagement was demonstrated in a series of biochemical assays evaluating PPARg binding and function.Cellbased assays confirmed that the compounds were cellularly active and modulate the function of PPARg.Antiproliferative activity was observed in a panel of cell lines selected based on their genetic dependency to PPARG deletion.
The combination of poor solubility, low permeability, and rapid hepatic clearance by GSH adduct formation from BAY-0069 resulted in a low exposure by all routes of administration.However, BAY-4931 and BAY-0069 show very robust in vitro effects in biochemical and cellular assays and provide novel tools to further the study of PPARg structure and function.nm.The purity of all target compounds was at least 95% as determined by UPLC-MS, with the exception of 6h (93%).Compound names were generated using ICS software.

2-(3-Methylphenyl
2-Amino-4-nitrophenol (1.80 g, 11.7 mmol) was dissolved in 90 toluene, then 1.0 eq.3-methylbenzoyl chloride (1.5 ml, 12 mmol) was added slowly.This mixture was refluxed for 20 hours.Then 0.25 eq.p-toluenesulfonic acid (555 mg, 2.92 mmol) was added and the mixture was refluxed for 6 more hours.The reaction mixture was allowed to cool to r.t..A dark precipitate had been formed which was collected by filtration and washed with toluene.The filtrate was evaporated under reduced pressure to give 3.54 g of the title compound (83% yield) as crude material which was used directly in the next step.
The organic phase was evaporated to dryness and the remaining crude material was purified by flash chromatography (silica gel, hexane/ ethyl acetate gradient) to give 200 mg (2.5% yield) of the title compound alongside 2.
Analytical SFC:  A mixture of 2-(4-ethylphenyl)-1,3-benzoxazol-5-amine (98.0 mg, 411 µmol), 1.0 eq.2-chloro-5-nitrobenzene-1-sulfonyl chloride (105 mg, 411 µmol), 1.1 eq.TEA (63 µl, 450 µmol) in 2.0 ml DCM was stirred at r.t. for 17 h.Water and DCM were added, and the phases separated.The aqueous phase was extracted multiple times with DCM.The combined organic phases were evaporated to dryness.The remaining crude material was purified by preparative HPLC (acidic conditions) to give 95.0 mg (50% yield, 100% purity) of the title compound 8f.Polyphoshoric acid (33 ml, 290 mmol) was heated 180 °C and 1.0 g 4-aminobenzoic acid (7.29 mmol) were added under vigorous stirring.The resulting mixture was stirred at 180 °C for 10 min and 1.0 g 2-amino-4,5-dimethylphenol (7.29 mmol) were added in portions.The resulting mixture was stirred at 180 °C for an additional 2 hours.The mixture was added to ice water.KOH was added and the pH-value adjust to pH: 10.The aqueous phase was extracted multiple times with DCM.The combined organic phases were evaporated to dryness to give 1.1 g crude material of the title compound which was used in the next step without further purification.UPLC-MS (Waters Acquity, acidic conditions)

UPLC-MS (Waters
A mixture of 4-(5,6-dimethyl-1,3-benzoxazol-2-yl)aniline (750 mg, 3.15 mmol), 1.3 eq.2-chloro-5-nitrobenzoic acid (825 mg, 4.09 mmol), 1.5 eq.HATU (1.80 g, 4.72 mmol) and 5.0 eq.TEA (2.2 ml, 16 mmol) in 14 ml DMF was stirred at r.t. until complete conversion.DCM and water were added, and the phases separated.The aqueous phase was extracted multiple times with DCM.The combined organic phases were evaporated to dryness and the remaining crude material purified by flash chromatography (silica gel, hexane/ ethyl acetate gradient) followed by preparative HPLC (acidic conditions) to give 190 mg ( 14% yield, 100% purity) of the title compound 8g.Water and DCM were added, and the phases separated.The aqueous phase was extracted multiple times with DCM.The combined organic phases were evaporated to dryness and the remaining crude material purified by flash chromatography (silice gel, hexane/ ethyl acetate gradient) to give 900 mg (70% yeld) of the title compound.A mixture of tert.-butyl2-chloro-5-(methylcarbamoyl)benzoate (900 mg, 3.34 mmol) and 8.3 ml HCl-solution (4N in dioxane, 10 eq.) was stirred at r.t. until complete conversion.DCM and water were added, and the phases separated.The aqueous phase was extracted multiple times with DCM.The aqueous phase was evaporated to dryness to give 700 mg (98% yield) of the title compound.
The organic phase was evaporated to dryness and the remaining crude material purified by preparative HPLC (acidic conditions) to give 14 mg (9% yield, 100% purity) of the title compound 9c.
UPLC-MS (Agilent, acidic conditions): Briefly, media was removed and cells were washed with PBS.Cells were fixed using 4% formaldehyde in PBS for 30 min at room temperature.The formaldehyde solution was removed and the cells were stained with crystal violet for another 30 min, after which the stain was removed and washed out.The plates were allowed to dry inverted overnight and imaged the following day using an Epson Perfection 600 scanner.
UM-UC-9 Proliferation: UM-UC-9 were grown in MEM alpha media (Gibco) containing 10% heat-inactivated fetal bovine serum (Sigma).The cells were stably transduced with a lentiviral expression vector encoding the TagGFP-Histone-2B protein (pTagGFP2-H2B, Evrogen).Cells were plated at 500 cells per well in a 384-well view-plate and allowed to attach at 37 C for 2 hours.Plates were dosed with compounds at indicated concentration in duplicate.7 days after addition of compound, nuclei were counted with the use of an In-cuCyte S3 Live Cell Imager.Cell counts were normalized to vehicle control and data reported as IC50, Emax and percent of vehicle control.All compounds reported were also evaluated in an independent experiment read out at day 5 with similar results.
FABP4 RTqPCR: High-throughput RTqPCR was performed using LightCycler 1536 (Roche) instrument according to the manufacturers protocol.RT112/84 cells were incubated the compounds for 24 hours and FABP4 (primer sequence: TAAACTGGTGGTGG AATGCG, GCGAACTTCAGTCCAGGTCA, TCATGAAA GGCGTCACTTCCACGAGA) expression was measured to monitor the activity of PPARG, RPL30 (primer sequence: GTCCCGCTCCTAAGGCAG, GTTGATCGACT CCAGCGACT, AGATGGTGGCCGCAAAGAAGACGAA) was used as a housekeeper gene.The qPCR was performed as a one-step measurement in cell lysates using LightCycler RNA Virus Master PCR Kit (Roche) according to the manufacturers protocol in a total volume of 1 µl.The data were first normalized to the housekeeping gene and are shown as relative expression compared to vehicle control.
In Vitro Metabolic Stability assay in Rat Hepatocytes: Generation of primary Hepatocyte suspension: Hepatocytes from male Wistar rats were isolated via a two-step perfusion method.After perfusion, the liver was carefully removed from the rat, the liver capsule was opened and the hepatocytes were gently shaken out into a Petri dish with ice-cold Williams' medium E (WME).The resulting cell suspension was filtered through sterile gauze into 50 mL Falcon tubes and centrifuged at 50 × g for 3 min at room temperature.The cell pellet was resuspended in 30 mL of WME and centrifuged through a Percoll gradient two times at 100 × g.The hepatocytes were washed again with WME and resuspend-ed in medium containing 5% FCS.Cell viability was determined by trypan blue exclusion.For the metabolic stability assays, liver cells were distributed in WME containing 5% FCS to glass vials at a density of 1.0 × 106 vital cells/mL.
The test compound was added to a final concentration of 1 µM.Organic solvent in the incubations was limited to ≤0.01%DMSO and ≤1% acetonitrile.During incubation, the hepatocyte suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45, and 90 min to which an equal volume of cold acetonitrile was immediately added.Samples were frozen at -20 °C overnight, and subsequently centrifuged at 3000 rpm for 15 min.The supernatant was analyzed with an Agilent 1200 HPLC system with MS/MS detection.The half-life of a test compound was determined from the concentration-time plot.From the halflife, the intrinsic and the in vitro predicted blood clearances were calculated as well as the hepatic extraction ratio EH = (CLb/LBF)•100%, according to the 'well-stirred' liver model 22 .In combination with the standardized liver blood flow (LBF) of 4.2 L/h/kg, a specific liver weight of 32 g/kg body weight and amount of liver cells in vivo (1.1 × 108 cells/g liver) and in vitro (1.0 × 106/mL) the in vitro blood clearance (CLb, in vitro) and the maximal bioavailability (Fmax in % = 1-EH*100%) were calculated.

In Vitro Metabolic Stability in Liver Microsomes:
The in vitro metabolic stability of test compounds was determined by incubation at 1 µM in a suspension of liver microsomes in 100 mM phosphate buffer pH 7.4 (NaH2PO4•H2O + Na2HPO4•2H2O) and at a protein concentration of 0.5 mg/mL at 37 °C.The microsomes were activated by adding a cofactor mix containing 8 mM glucose-6-phosphate, 0.5 mM NADP, and 1 IU/mL glucose-6-phos¬phate dehydrogenase in phosphate buffer pH 7.4.The metabolic assay was started shortly afterwards by adding the test compound to the incubation at a final volume of 1 mL.During incubation, the microsomal suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45, and 60 min.Further handling and analysis as per the hepatocyte method described above with the human specific scaling factors of a liver blood flow of 1.32 L/h/kg and a specific liver weight of 21 g/kg body weight.

Estimation of Plasma Protein Binding by Flux Dialysis:
Binding of test compounds to plasma proteins is measured by a modification of standard equilibrium dialysis in a 96well format using HT-Dialysis equipment made of Teflon at 37°C and 5% CO2 atmosphere.The Flux dialysis method is based on the principle that the initial flux rate (R_slope) of a compound is proportional to the product of compound initial concentration, fu and unbound dialysis membrane permeability (P_mem).Therefore, fu can be determined from R_slope when membrane P_mem is known.Common equipment and assay specific P_mem of 75.2x10-6cm/s which was established was used for calculation 23 .
In brief, a semipermeable membrane (regenerated cellulose, MWCO 12-14K) separates the plasma donor and plasma receiver side filled with 150 µl plasma each.The test compound is added to the donor side at 1 µM and binds to plasma proteins.The unbound fraction of the test compound passes the membrane and distributes on both sides until equilibrium is reached.The flux rate as the rate of compound appearance into the receiver side is approximated from the time course of the quotient of receiver and donor concentration (R) by non-linear regression including data from an entire time course.For this purpose, samples are taken at different time points (up to 96 h) from the donor and receiver side and the relative compound concentration (peak area ratios analyte/IS) is measured by LC-MSMS analytics.Prior to this both sides are matrix matched (diluted with buffer and plasma to achieve the same matrix of 10% plasma) and subsequently precipitated with a fourfold volume of methanol containing an appropriate internal standard (IS).
The bidirectional transport assay for the evaluation of Caco-2 permeability was undertaken in 24-well insert plates using a robotic system (Tecan).Before the assay was run, the culture medium was replaced by transport medium (FCS-free HEPES carbonate transport buffer pH 7.2).For the assessment of monolayer integrity, the transepithelial electrical resistance (TEER) was measured.Only monolayers with a TEER of at least 400 Ω*cm2 were used.Test compounds were predissolved in DMSO and added either to the apical or basolateral compartment at a final concentration of 2 µM.Evaluation was done in triplicate.Before and after incubation for 2 h at 37 °C, samples were taken from both compartments and analyzed, after precipitation with MeOH, by LC-MS/MS.The apparent permeability coefficient (Papp) was calculated both for the apical to basolateral (A→B) and the basolateral to apical (B→A) direction using following equation: Papp = (Vr/P0)(1/S)(P2/t), where Vr is the volume of medium in the receiver chamber, P0 is the measured peak area of the test compound in the donor chamber at t = 0, S is the surface area of the monolayer, P2 is the measured peak area of the test compound in the acceptor chamber after incubation for 2 h, and t is the incubation time.The efflux ratio basolateral (B) to apical (A) was calculated by dividing Papp B-A by Papp A-B.
Metabolite Identification in rat Hepatocytes: The test compound was incubated at 37 °C in a hepatocyte suspension in a round-shaking water bath at 116 rpm containing 1x10 6 cells/ mL for 1, 2 and 4 h. 5 µM test compound was added from a 0.1 mM stock solution dissolved in acetonitrile.Enzymatic activities of all hepatocyte preparations were measured using a variety of standard substrates.All hepatocytes exhibited good activities.The incubations were terminated by the addition of acetonitrile (approx.30% (v/v)) and stored at 18 °C until analysis.Prior to analysis the samples were thawed and centrifuged at 12000 rpm for 10 min.Aliquots of 10 µL of the supernatant were used to control the recovery of radioactivity by liquid scintillation counting.Aliquots of the supernatants were transferred into HPLC vials and analyzed by HPLC and on-line MS detection using the Orbitrap Fusion Lumos mass spectrometer and parallel split to UV detection.Exact mass and mass changes in comparison with the parent drug in combination with the fragment pattern from MS/MS were used to propose the structures of metabolites or confirm structures with that of reference compounds.

CYP Inhibition:
The inhibitory potency of test compounds towards CYP450-dependent metabolic pathways was determined in pooled human liver microsomes (Xenotech, USA) by applying individual CYP isoform selective standard probes (CYP1A2: phenacetin; CYP2C8: amodiaquine; CYP2C9: diclofenac; CYP2D6: dextromethorphan; CYP3A4: midazolam).Reference inhibitors were included as positive controls.Incubation conditions (protein and substrate concentration, incubation time) were optimized regarding linearity of metabolite formation.Assays were processed in 96-well microtiter plates at 37 °C using a Genesis Workstation (Tecan, Crailsheim, Germany).After protein precipitation, metabolite formation was quantified by LC-MS/MS analysis, followed by inhibition evaluation and IC50 calculation.
PXR NOEL Assay: A HepG2 cell line stably-cotransfected with a vector for human PXR and a Luciferase reporter gene under the control of a human CYP3A4 promotor were seeded in a 384 well plate and cultivated at 37 °C/5% CO2 in humidified air.24h prior read-out the cells were treated with compound in a ten-step serial dilution of 1:3 starting at the highest test concentration of 50 µM and ending at 2 nM.Rifampicin was incubated in the same manner as positive control.In addition, for the normalization of the luminescence signal cells were incubated with Rifampicin at a concentration of 16.7 µM corresponding to 100% activation, as well as DMSO for background luminescence corresponding to 0% activation (n=32 wells each).Cells were lysed and incubated with the Luciferase substrate ONE-Glo™ Reagent (Promega, Madison WI, USA) according to manufacturer's instructions and luminescence signal was detected in a plate reader.A concentration-dependent increase of the luciferase activity above 10% of Rifampicin control was classified as PXR transactivation.
In vivo exposure: All animal experiments were conducted in accordance with the German Animal Welfare Law and were approved by local authorities.Female NMRI nu/nu mice (Janvier, France) were dosed once at 100 mg/kg orally (p.o., in PEG400/Ethanol 9:1) (n=3 per group), intraperitoneally (i.p., in Solutol/Ethanol/Water 4:1:5), or subcutaneously (s.c. in castoroil/benzylbenzoate 9:1).After 0.5h, 3h, 6h, 14h, 24h and 48h mice were sacrificed by decapitation and blood sampled in Potassium-EDTA tubes (Sarstedt, Germany).100 µL plasma were used for analysis.Samples were precipitated by immediate administration of ice cold acetonitrile in 1:5 dilution.Samples were frozen at -20 °C overnight, and subsequently centrifuged at 3000 rpm for 15 min.The supernatant was analyzed with an Agilent 1200 HPLC system with MS/MS detection (AB Sciex, Framingham, MA, USA).Obtained exposures were corrected for plasma protein binding and put into relation to the antiproliferative IC50u.

Structural Biology
Production of recombinant PPARg: Codon optimized DNA sequences of human PPARG ligand binding domain (LBD), including residues 231-505 (Uniprot: P37231-1/Isoform 2), were synthesized (Gene Art, Life Technologies) and inserted into a modified pET22b vector for the overexpression of His-fusion proteins in Escherichia coli.All plasmids contained a thrombin protease cleavage site between the N-terminal 6xHis tag and the protein of interest.The protein was overexpressed in Escherichia coli BL21(DE3) overnight at 17°C after induction by IPTG.
In brief, cell pellets were resuspended in Lysis Buffer (20 mM TRIS pH 8.0, 150 mM NaCl, 1 mM DTT, and 20 mM imidazole with EDTA complete (Roche Applied Science)) and mechanically lysed using a Microfluidizer.Lysates were centrifuged at 30,000×g for 1 h, and clear supernatants were loaded on a 5 mL Protino Ni-NTA FPLC column (Macherey-Nagel GmbH).Bound protein was washed with Buffer A (20 mM TRIS pH 8.0, 150 mM NaCl, 1 mM DTT, and 20 mM imidazole) and high-salt Buffer A (300 mm NaCl).The His-/fusion-tag was removed by thrombin cleavage on the column overnight at 16°C.Cleaved PPARg LDB and thrombin were eluted with Buffer A. The final step of purification included addition of Benzamidine Sepharose (Cytiva) to remove thrombin and size exclusion chromatography (10 mM TRIS pH 8.0, 100 mM NaCl, 5 mM DTT, and 1 mM EDTA).Purified protein was concentrated and stored at -80 °C.
Crystallization and structure determination: Purified human PPARg LBD (9-12 mg/mL; frozen stock) was incubated with the peptide and ligand at the indicated protein-to-peptide-toligand molar ratios (Table S1) for 1-5 h at room temperature.Small-molecule ligands were dissolved to 100 mM in DMSO and further diluted to 10 mM in ethanol.The NCOR2-ID2 peptide (H2343-W2365: HASTNMGLEAIIRKALMGKYDQW) was purchased (Biosyntan GmbH, Germany) and dissolved without further purification to 10 mM in protein buffer.All crystallization experiments were performed at 20°C as sitting drops by adding equal volumes of sample and reservoir solution (100-300 nL).Crystallization conditions are listed in Supplementary Table 1.Crystals were frozen in liquid nitrogen after cryoprotection with 25% glycerol.
Diffraction data was collected at the Proxima-2 beamline (Soleil synchrotron, Paris, France) and was processed with XDS 24 .See Supplementary Table 1 for data and refinement statistics.The structures were solved using molecular replacement as implemented in Phaser and Dimple 25 .Further refinement of the initial models was accomplished through multiple rounds of refinement in Refmac5 25 and manual fitting and re-building in Coot 27 .Restraints for small-molecule ligands were generated with ProDrg 28 and modified for covalent linkage to a cysteine residue with JLigand 29 .

Figure 2
Figure 2 Discovery of PPARg inverse-agonists.A. Representation of hit finding screening cascade from ultrahigh-throughput competitive ligand-binding assay of 4.3 million compounds, single

Figure 3 .Scheme 1 .
Figure 3. Crystal structure of PPARg bound to NCOR2 co-repressor peptide and the inverse-agonist compound 6a (PDB ID: 8AQM).A. Left: Insert showing the complete trimeric complex with the views of the different panels indicated by lines and arrows.Right: Overview of the PPARg (green) co-complex with the C-terminal Helix-12 (red) bound behind the kinked Helix-3, NCOR2 peptide (magenta), and compound 6a (cyan) covalently bound to Cys313.The very C-terminus, Tyr505, interacts directly with 6a.B. Intramolecular interactions of Helix-12 when sequestered into the canonical ligand binding site.C. NCOR2 co-repressor binding interactions to PPARg.SAR: Initial efforts focused on improving the interactions between compounds and NCOR2.Compounds with variable NCOR-interacting rings were synthesized according to Scheme 1. Synthesis of the necessary anilines was achieved by either reduction of the corresponding nitro-arene under SnCl2 conditions or via a Buchwald-Hartwig coupling of the

Figure 4 .
Figure 4. Structural details of the interactions between PPARg /NCOR2 and 6a (PDB ID: 8AQN) and the optimized inverse-agonists BAY-4931 (PDB ID: 8AQN).A. Co-crystal structure of PPARg (green), NCOR2 peptide (magenta), and 6a (cyan).Polar interactions are highlighted with yellow dashes.B. Co-crystal structure of PPARg (green), NCOR2 peptide (magenta), and BAY-4931 (yellow).C. Comparison of the crystal structures with 6a (gray) and BAY-4931 (colored as in B).A water-mediated interaction between BAY-4931, Gln314, and Asn2347 of the NCOR2 peptide is highlighted.The additional hydrophobic interaction of the para ethyl group of BAY-4931 is indicated by a black arrow (lower left).The increased Emax observed in the corepressor recruitment assay by para-substituted compounds may be explained by increased and/or stabilized interactions with the NCOR2 peptide.To test this hypothesis, BAY-4931 was co-crystallized with PPARg (Fig 4).The covalently bound inverse-agonist, 6a, extends towards the NCOR2 peptide and is located slightly underneath the N-terminus of the co-repressor (Fig 4A).Para-substituted BAY-4931 shows hints of stabilized recruitment of the NCOR2 peptide (Fig 4B).The NCOR2 peptide exhibits a modest shift in position and two additional N-terminal residues of the peptide could be modeled with confidence.The introduced ethyl substitution fits well into a niche formed between the receptor and the co-repressor.Also, with BAY-4931, a more extensive water-network was observed, now extending all the way from PPARg /BAY-4931 to the NCOR2 peptide.Comparison of the two complexes otherwise show minimal changes in ligand binding mode and protein conformations (Fig 4C).Extending the Nterminus of the NCOR2 peptide did not change any of the observed interactions or increase the number of resolved residues.Taken together, this is in line with the biochemical and cellular data of the two compounds (Table 1) showing

Figure 5 .
Figure 5. Identification of compound 6a metabolic reaction products by incubation with rat hepatocytes.UV chromatogram of 6a and its metabolite P-1 before (blue) and after 1 h incubation (red) in rat hepatocytes and structure proposal of the metabolite P-1 based on the exact masses of the metabolite P-1 and its MS-MS fragments

Figure 6 .
Figure 6.BAY-4931 and BAY-0069 are selective inverse-agonists of PPARg.Biochemical TR-FRET evaluation of ligand-dependent changes in the interaction between PPARg ligand-binding domain and interacting peptide fragments from A. co-activator MED1, B. co-repressor NCOR1, and C. co-repressor NCOR2.D. Dose-response effects of compound on the proliferation of UMUC9-H2B-GFP cells.E. Dose-response effects of compound on reporter activity in RT112-FABP4-NLucP F. Crystal violet colony formation assays comparing effects of PPARg modulators with bladder cell lines highlighted in blue and pancreatic in orange.Samples in triplicate, representative well shown.See supporting information for additional data.

Figure 8 .
Figure 8. Pharmacodynamic regulation of PPARg target gene expression in UM-UC-9 xenograft bearing mice.Expression of FABP4 and ANXA3 normalized to housekeeping gene, PPIA, and reported as normalized expression relative to vehicle control.Mice were treated as indicated and 3 -4 were sacrificed at the respective time points.Statistical analysis: one-way ANOVA with Dunnett's multiple testing correction; *: p ≤ 0.05; **: p ≤ 0.01.

Table 2 : ADME of selected NCOR2 interacting ring modifications
a) Human microsomal stability determined by the incubation of 1 µM of compound with human liver microsomes for 1 h.Refer to Experimental Section for assay conditions.Clearance value represents one experiment.b) Rat hepatocyte stability assay determined by the incubation of 1 µM of compound with rat liver hepatocytes for 1.5 h.Refer to Experimental Section for assay conditions.Clearance value represents one experiment.c) Compound stability in buffer containing 500 µM of glutathione.Refer to Experimental Section for assay conditions.% recovery represents one experiment.d) Compound stability in buffer containing 500 µM of cysteine.Refer to Experimental Section for assay conditions.% recovery represents one experiment.e) Caco-2 permeability assay.Refer to Experimental Section for assay conditions.Permeability and efflux ratio represent a single experiment.f) Thermodynamic solubility of compound in pH 6.5 PBS buffer from a DMSO stock.Refer to Experimental Section for assay conditions.

Table 4 : Tier 1 ADME of selected compounds from Table 3
a) Human microsomal stability determined by the incubation of 1 µM of compound with human liver microsomes for 1 h.Refer to Experimental Section for assay conditions.Clearance value represents one experiment.b) Rat hepatocyte stability assay determined by the incubation of 1 µM of compound with rat liver hepatocytes for 1 h.Refer to Experimental Section for assay conditions.Clearance value represents one experiment.c) Compound stability in buffer containing 500 µM of glutathione.Refer to Experimental Section for assay conditions.% Recovery represents one experiment.d) Compound stability in buffer containing 500 µM of cysteine.Refer to \ Experimental Section for assay conditions.% Recovery represents one experiment.e) Caco-2 permeability assay.Refer to Experimental Section for assay conditions.Permeability and efflux ration represent a single experiment.f) Thermodynamic solubility of compound in pH 6.5 PBS buffer from a DMSO stock.Refer to Experimental Section for assay conditions.

Table 7 : Selectivity and metabolic liability profile of BAY-4931 and BAY-0069
IC50 (nM) and Emax (% inhibition of luciferase reporter activity) or activation (+) values are from one experiment with a 10 point dose-response curve with 4 replicates per point for a) mouse Pparg, b) human PPARg, c)PPARA, and d) PPARD.e-i) IC50 (µM) values for CYP inhibition assays with specific isoform indicated.Data represent the mean from one experiment performed in duplicate.j) PXR (NR1I3) no effect level IC50 (µM).